Environmental stresses such as UV irradiation, oxidative damage, heat shock, and exposure to arsenic or heavy metals induce expression of genes that alleviate cellular injury. An important contributor to this stress response is a family of protein kinases that phosphorylate the a subunit of eukaryotic initiation factor -2 (elF2), leading to regulation of general protein synthesis and gene-specific translation. The focus of this grant is to understand the mechanisms by which phosphorylation of elF2a induces translation of certain mRNAs that contain ORFs in their 5'-leader sequences. This issue will be addressed by studying activating transcription factor-4, ATF4 that is central to the elF2a kinase stress response. ATF4 is important for inducing over fifty genes involved in metabolism, the cellular redox status, apoptosis, and additional transcriptional factors, ATF3 and CHOP/GADD153. We have shown using luciferase reporter constructs that the 5'-leader of the ATF4 mRNA has two upstream ORFs that differentially regulate ATF4 translation in response to elF2a phosphorylation. We propose a model whereby the 5'-proximal uORF1 is a positiveacting element that facilitates ribosome scanning and reinitiation at downstream ATF4 mRNA coding region. In Aim1, we will test this differential ribosome reinitiation model in vitro by developing a reticulocyte lysate translation system that induces gene-specific translation in response to elF2a phosphorylation. This in vitro translation system will be then be adapted to study ribosome mapping along the ATF4 mRNA by the toeprinting assay. In Aim2, we will utilize the developed in vivo and in vitro methods to characterize additional genes that are subjected to translational control such as the oncogene MDM2 and the transcription factor ATF3. Together these studies will be important for understanding the molecular mechanisms regulating gene specific translation in higher eukaryotes during adaptive responses to environmental stresses.